Project Summary/Abstract Multiple myeloma (MM) remains an incurable malignancy for which the development of new therapeutic approaches is required. The therapeutic agents to be developed in this project are fusion proteins with type I or type II interferon (IFN) genetically fused to an antibody that recognizes the CD138 antigen present on MM cells. IFNs have potent anti-tumor properties and the potential to be effective cancer therapeutics. Indeed IFN? has been used in the treatment of MM, but its clinical use has been limited because it is difficult if not impossible to achieve therapeutically effective doses at the site of the tumor without causing systemic toxicity. We hypothesize that IFN fused to an antibody that recognizes CD138 will be selectively targeted to the tumor and will be able to achieve an effective concentration without being toxic. Our experiments to date support this hypothesis. One objective of the current undertaking is to develop and characterize the in vitro and in vivo effectiveness of fusion proteins (FPs) containing IFN?2, IFN?14, a mutant of IFN?2 (IFN?2YNS) with high affinity for the IFN receptor, or IFN?, with the goal of identifying the optimal therapeutic for MM. The proteasome inhibitor bortezomib and the thalidomide derivative lenalidomide are FDA-approved therapies effective in treating but not curing MM. We will now determine if combining the most efficacious FPs with these standard therapies will result in additivity or synergy of the in vitro and in vivo anti-tumor effects. MM tumors consist not only of plasma cells and plasmablasts, but also of subpopulations of pre-plasmablasts and CD20+ B cell progenitors. In an effort to fully eradicate all MM cells, we will now test the hypothesis that combining anti-CD20-IFN FPs with anti-CD138-IFN FPs will yield improved efficacy in mice bearing MM xenografts. One important property of the IFNs is their ability to potentiate anti-tumor immune responses. To study FP therapy in a physiologically-relevant system that mimics human MM, including the role of the bone marrow microenvironment, we will use two well-characterized models, the V?*MYC and the Bcl-XL/Myc cell lines, and will target murine CD138. Modeling of this therapeutic approach in immunocompetent mice will allow several important variables to be explored, including the responsible immune effector mechanisms, possible immune- related toxicities, and the potential for secondary ?in situ vaccination? effects. The role of host IFN receptors will be evaluated using the V?*MYC model and IFN receptor deficient C57BL/6 mice. The role of the IFN receptors expressed on the MM cells will be evaluated by inhibiting their expression on 589 cells using RNAi. Using these models, the potential synergy between FPs and bortezomib or lenalidomide in preventing tumor growth will be determined. The ability of immune checkpoint blockade to enhance FP efficacy will also be evaluated using treatment with anti-PD-1/L1. We believe that successful completion of these studies may provide the data to support the translation of anti-CD138 fusion proteins into the clinic.